Abstract
Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared with noninduced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wild-type EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the colocalization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase and, in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction.
Highlights
That express the EWSR1/FLI1 fusion gene, while the remaining patients have tumors that express fusion genes composed of EWSR1 and other ETS transcription factors (ETV1, ETV4, ERG, and FEV1) [4,5,6,7,8]
Because our previous study showed that the N terminus of EWSR1/FLI1 is required to induce mitotic defects, we used a candidate approach to investigate the critical residues of the fusion protein [30]
We investigated residue Thr 79 of EWSR1/FLI1, which is located in the N terminus of the molecule, because it is a known site for posttranslational modifications
Summary
EWSR1/FLI1 localizes to the chromosomes at prophase, and residue Thr 79 is critical for this localization. The majority of cells expressing EWSR1/FLI1 (Dox+) had nondiploid chromosome counts (Fig. 5D) These results suggest that EWSR1/ FLI1 induces aneuploidy and that the Thr 79 residue of EWSR1/FLI1 plays a critical role in this defect. The EWSR1/ FLI1 and EWSR1/FLI1-T79D expressing DLD-1 cell lines were subjected to immunocytochemistry using anti-mCherry and Alexa Fluor 594 antibodies and anti-Aurora B and Alexa Fluor 488 antibodies, followed by the same colocalization analysis as Figure 3 using the Pixelmap function of Fiji Imaging software. There was no significant difference between the incidence of aberrant Aurora B localization in EWSR1/FLI1expressing cells and EWSR1/FLI1-T79D-expressing cells (p > 0.05, nonsignificant) These results suggest that phosphorylation of the Thr 79 residue of EWSR1/FLI1 is critical for the EWSR1/FLI1-induced mislocalization of Aurora B at the midzone during anaphase. Our results suggest these phenotypes are mediated through a single pathway: the localization of EWSR1/FLI1 to the chromosomes during prophase and metaphase triggers defects in the midzone and leads to the induction of aneuploidy (Fig. 10)
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